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Examining the persistence of human Coronavirus 229E on fresh produce

Madeleine Blondin-Brosseau, Jennifer Harlow, Tanushka Doctor, Neda Nasheri

PII: S0740-0020(21)00045-9
DOI: https://doi.org/10.1016/j.fm.2021.103780
Reference: YFMIC 103780

To appear in: Food Microbiology

Received Date: 4 December 2020

Revised Date: 24 February 2021
Accepted Date: 24 February 2021

Please cite this article as: Blondin-Brosseau, M., Harlow, J., Doctor, T., Nasheri, N., Examining the
persistence of human Coronavirus 229E on fresh produce, Food Microbiology (2021), doi: https://
doi.org/10.1016/j.fm.2021.103780.

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1 Examining the Persistence of Human Coronavirus 229E on Fresh

2 Produce

3 Madeleine Blondin-Brosseau1, Jennifer Harlow1, Tanushka Doctor1, and Neda Nasheri1, 2

4 1- National Food Virology Reference Centre, Bureau of Microbial Hazards, Health Canada,
5 Ottawa, ON, Canada
6 2- Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine,

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7 University of Ottawa, ON, Canada

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9 Corresponding author: Neda Nasheri neda.nasheri@canada.ca

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12 Abstract

13 Human coronaviruses (HCoVs) are mainly associated with respiratory infections. However, there

14 is evidence that highly pathogenic HCoVs, including severe acute respiratory syndrome

15 coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome (MERS-CoV), infect the

16 gastrointestinal (GI) tract and are shed in the fecal matter of the infected individuals. These

17 observations have raised questions regarding the possibility of fecal-oral route as well as

18 foodborne transmission of SARS-CoV-2 and MERS-CoV. Studies regarding the survival of

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19 HCoVs on inanimate surfaces demonstrate that these viruses can remain infectious for hours to

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20 days, however, there is limited data regarding the viral survival on fresh produce, which is

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usually consumed raw or with minimal heat processing. To address this knowledge gap, we
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22 examined the persistence of HCoV-229E, as a surrogate for highly pathogenic HCoVs, on the
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23 surface of commonly consumed fresh produce, including: apples, tomatoes, cucumbers and
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24 lettuce. Herein, we demonstrated that viral infectivity declines within a few hours post-

25 inoculation (p.i) on apples and tomatoes, and no infectious virus was detected at 24h p.i, while
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26 the virus persists in infectious form for 72h p.i on cucumbers and lettuce. The stability of viral
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27 RNA was examined by droplet-digital RT-PCR (ddRT-PCR), and it was observed that there is

28 no considerable reduction in viral RNA within 72h p.i.

29 Keywords

30 Human coronavirus, persistence, fecal-oral transmission, foodborne transmission, plaque assay,

31 droplet-digital RT-PCR, decay rate

32
33 Introduction

34 Coronaviruses that infect humans (HCoV) belong to alpha and beta genera of the Coronaviridae

35 family. Four common HCoVs (229E, OC43, HKU1, and NL63) are responsible for 10-30% of

36 common cold symptoms that can be mild to moderate (Perlman and Netland. 2009). SARS-CoV-

37 2, which is responsible for the coronavirus disease 2019 (COVID-19) pandemic, is a

38 betacoronavirus that uses angiotensin conversion enzyme 2 (ACE-2) for entry into the host cells.

39 ACE-2 is abundantly expressed in the epithelium of the respiratory tract as well as the oral

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40 cavity, intestine and colon (Lamers, et al. 2020, Qian, et al. 2020). It is evident now that a

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41 considerable proportion of COVID-19 patients demonstrate gastrointestinal symptoms including

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nausea, vomiting, diarrhea, and abdominal pain (Cheung, et al. 2020, Zhou, et al. 2020,
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43 Scaldaferri, et al. 2020). SARS-CoV-2 RNA has been detected in more than 50% of patients’
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44 stool specimens (Wolfel, et al. 2020, Wang, et al. 2020, Huang, et al. 2020, Cha, et al. 2020), and
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45 several studies have confirmed that the virus detected in stool is infectious (Xiao, et al. 2020,

46 Zhou, et al. 2020). Moreover, persistent fecal viral shedding has been observed in pediatric
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47 patients (Xu, et al. 2020) and there is evidence that SARS-CoV-2 can replicate productively in
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48 human enteroids and enterocytes (Lamers, et al. 2020, Zhou, et al. 2020). More recently, it was

49 demonstrated that multi-route mucosal inoculation (including oral inoculation) of African green

50 monkeys with SARS-CoV-2 results in infection in both the respiratory and gastrointestinal tract

51 (Hartman, et al. 2020), and orally inoculated golden Syrian hamsters develop respiratory and

52 intestinal infection (Chak-Yiu Lee, et al. 2020). Collectively, these observations suggest that

53 fecal-oral transmission of SARS-CoV-2 is possible.

54 Although the primary route of transmission for HCoVs is inhalation of contaminated respiratory

55 droplets and possible direct contact with contaminated fomites, there is concern that food could
56 also act as a vehicle of transmission if contaminated with HCoVs. Food may become

57 contaminated with HCoVs by contact with body secretions or fluids or by contact with soiled

58 hands. Also, HCoVs may become aerosolized via talking, sneezing, or coughing of food handlers

59 and then be deposited on food surfaces. Food not only may act as a fomite, but can also transport

60 the virus to the potentially susceptible oral cavity and the GI tract (Xu, et al. 2020). There is

61 evidence that certain HCoVs including HCoV-229E and MERS can survive GI conditions

62 including low pH, digestive enzymes and bile (Zhou, et al. 2017). If this is the case for SARS-

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63 CoV-2, the relatively high viral titre in stool and rectal swabs of the infected individuals could be

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64 explained by active viral replication in the GI tract. Furthermore, fecal-oral is the main route of

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transmission for enteric coronaviruses such as swine coronaviruses (Wang, et al. 2019), canine
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66 coronaviruses (Decaro and Buonavoglia. 2011), and equine coronavirus (Pusterla, et al. 2018) ,
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67 demonstrating that these viruses are not sensitive to the GI fluids.

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68 Although the results should be cautiously interpreted, China has reported the finding of SARS-
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69 CoV-2 in imported frozen food commodities (Roxanne, 2020; Yusha, 2020), and it was shown
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70 that the isolated virus from imported frozen cod is infectious in tissue culture (Liu, et al. 2020).

71 More recently, genetic evidence was provided that would link COVID-19 resurgence in Beijing

72 to cold-chain food contamination (Pang, et al. 2020). It was also demonstrated that this virus is

73 stable for weeks in cold storage (-80°C to +4°C) on artificially contaminated pork, chicken and

74 salmon (Fisher, et al. 2020). However, there is limited data on HCoVs survival on fresh produce.

75 Contamination of fresh produce may result in the transmission of not only the enteric viruses that

76 are traditionally considered foodborne pathogens, but also possibly respiratory viruses such as

77 adenoviruses, coronaviruses, and influenza viruses that can infect via contact with mucosal

78 membranes (O'Brien, et al. 2020). This is of particular concern for uncooked fruits and
 79 vegetables. Additionally, food handlers infected with respiratory viruses could potentially

80 contaminate “cold foods” such as salads and sandwiches (Yepiz-Gomez, et al. 2013), and spread

81 the infection through various routes such as close contact and fomites. Thus, it is imperative to

82 examine the viral behaviour and inactivation on fresh produce.

83 Since working with SARS-CoV-2 requires biosafety level 3 (BSL-3) laboratory containment

84 conditions, the use of surrogate HCoVs have been suggested to expand the current knowledge on

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85 coronavirus survival and inactivation under various conditions (Guillier, et al. 2020). For this

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86 reason, we chose HCoV-229E as a surrogate virus, since it has similar physicochemical

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properties to the more virulent HCoVs responsible for MERS and SARS (Warnes, et al. 2015).

88 In this study, we examined the ability of HCoV-229E to retain infectivity on the surface of select
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89 fruits and vegetables, and thus obtained representative survival data that can be used to conduct
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90 risk assessments of SARS-CoV-2 transmission via food.

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91 Materials and Methods

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92 Cells and Viruses:

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93 HCoV-229E and human embryonic lung cell line MRC-5 were obtained from the American

94 Type Culture Collection (CCL-171 and VR-740, respectively). Cells were grown at 37°C and

95 5% CO2 in culture media composed of Eagle’s minimal essential medium, supplemented with

96 0.23% (w/v) sodium bicarbonate, 500 µg/mL Penicillin-Streptomycin (ThermoFisher scientific),

97 1% Glutamax-1, 1% non-essential amino acids, and foetal bovine serum (FBS) 5% (v/v).

98 -Sample preparation:

99 Four different produce types, all purchased from local grocery stores in Ottawa, Ontario, were

100 tested: Royal Gala apples, Traditional Series tomatoes, English cucumbers, and Romaine lettuce
101 (PLU code 4173, 4799, 4593, and 4640, respectively). Ten time points were selected, in

102 triplicates: 0h, 0.5h, 1h, 2h, 4h, 6h, 16h, 24h, 48h and 72h. For Romaine lettuce, only 4 time

103 points were tested: 0h, 24, 48h and 72h. Each of the produce items was rinsed with water, dried

104 with Kimwipes and disinfected with 70% ethanol. On the surface of each produce item, a 5cm by

105 5cm square was delimited using tape. For Romaine lettuce, the square was carefully drawn on

106 each leaf to include both the rib and the leafy part. This area was inoculated with 100µL of

107 HCoV-229E (ATCC VR-740, 5×105 PFU/mL). The liquid was spread using the tip of the

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108 pipette, then allowed to fully dry for 1h. After the appropriate time lapse at ambient conditions

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109 (22°C; relative humidity, 30% to 40%), the surface was sampled with a cotton swab, according

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to the ISO/TS 15216-1:2017 (ISO. 2017) method, which was then placed into the MRC-5 culture
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111 media previously described (Nasheri, et al. 2020). Samples were processed immediately after
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112 swabbing.
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113 Viral quantification:

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114 - plaque assay:

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115 Viral quantification and survival time were determined by plaque assay using MRC-5 cells. Cells

116 were grown at 37 °C and 5% CO2 in the culture medium previously described for up to three

117 days, before being seeded, transferred into 12-well plates at a targeted concentration of 5×105

118 cells/mL and incubated to reach a confluency of 80-90%. Samples were diluted in culture

119 medium and 100µL of at least two dilutions were used in duplicate to infect the prepared plates

120 for 90 min at 35°C and 5% CO2. Plates were manually rocked every 10 min during the infection

121 phase. Cells were then washed with phosphate buffered saline (PBS) and covered with 2mL of

122 overlay media, composed of a 50/50 mix of 2× culture medium previously described and 0.5%
123 agarose. Plates were incubated at 35°C and 5% CO2 for 3-4 days. Cell monolayers were fixed

124 using 3.7% paraformaldehyde for 4-16 h, freed from overlay plugs by running under tap water

125 and stained with 0.1% crystal violet for 20 min. Plaques were counted for each dilution to

126 determine the viral titre.

127 - Determining limit of detection

128 Each produce item was artificially inoculated with a serial dilution of the viral stock in triplicate.

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129 At T0, the virus was extracted and assayed by plaque assay as described above. The plaques were

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130 counted for each dilution and results were analyzed to determine the highest dilutions (lowest

131
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titre) for which plaques were still obtained in triplicate experiments.
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132 - Recovery rate calculation
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133 The recovery efficiency was determined by calculating the ratio between the viral titre recovered
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134 at T0 and the viral titre that was used to inoculate the sample.
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135 Recovery rate (%): × 100
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136 - Estimating the decay rate:

137 Viral decay rate was calculated as described previously (Long and Short. 2016). Briefly, linear

138 regressions of the natural logarithm of virus abundance versus time (in hours) was calculated.

139 The slope of the regressions represent the decay rate and when multiplied by 100, represent

140 percentage of infectivity lost per hour. Viral half-life was calculated by dividing ln(2) by the

141 slope.

142 - ddRT-PCR:
143 For each produce item, except lettuce, all triplicates of 10 time points were tested. Viral RNA

144 was isolated using a QIAamp viral RNA kit (QIAGEN) and diluted in sterile molecular biology

145 grade water (Corning). The QX200 ddPCR system (Bio-Rad) was used for quantification and all

146 PCR reactions were prepared using the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad

147 Cat# 1864022). Primers used were previously described in (Vijgen, et al. 2005): Forward primer

148 229E-FP (5-TTCCGACGTGCTCGAACTTT-3; GenBank accession no. M33560; nt 474 to 493)

149 and reverse primer 229E-RP (5-CCAACACGGTTGTGACAGTGA-3; nt 523 to 543). A new

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150 probe that would complement the primers and be compatible with TaqMan qPCR requirements

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151 (ABI 7700 Users Manual) was designed by using Integrated DNA Technologies (IDT)

152
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OligoAnalyzer tool. The new probe had the appropriate dissociation temperature and a minimal
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153 likelihood for duplex or hairpin formation: 229E-PR (5’-/56-
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154 FAM/TGCATTGAC/ZEN/CTCAGGATTCCATGCCC/3IABkFQ/-3’). Each PCR reaction

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155 contained 5µL of RNA, 1000 nmol/L of each primer, and 280 nmol/L of each probe. All samples

156 were tested in duplicate. Droplets were generated using the QX200 droplet generator (Bio-Rad)
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157 according to the manufacturer’s protocols, and PCR was performed using the following cycling
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158 conditions: an initial reverse transcription at 48°C for 30 min, followed by PCR activation at

159 95°C for 10 min and 45 cycles of amplification (15 s at 95°C and 1 min at 60°C). Droplets were

160 detected in the QX200 droplet reader and analyzed using the Quantasoft version 1.7.4.0917 (Bio-

161 Rad) software.

162 Results

163 Recovery Efficiency from Produce

164 As shown in Table 1, the recovery efficiency for infectious HCoV-229E from all the tested

165 commodities is well above 1%, with the highest recovery rate (10.8%) from tomatoes and the
166 lowest (4.1%) from cucumbers. The limit of detection (LOD) for each commodity is determined

167 as the lowest spiking concentration that produced plaques for all three replicates. As indicated in

168 Table 2, the LOD was approximately 125 PFU for tomatoes and apples, and 50 PFU for

169 cucumbers.

170 Persistence of infectivity

171 We artificially inoculated the surface of apples, tomatoes and cucumbers with 5×104 PFU of

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172 HCoV-229E, which is consistent with the amount of virus that is typically exhaled by an infected

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173 individual (Ma, et al. 2020). Figure 1 shows the persistence in infectivity of HCoV-229E at RT

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within 72 h p.i. The change in infectious viral titre is similar in apples and tomatoes with a

175 progressive decline in infectivity up to 16h p.i. (Figure 1, Table 3). No infectious viral particles
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176 were isolated from tomatoes and apples at 24 h p.i., which demonstrates that viral infectivity is
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177 reduced below the LOD (i.e. >3 log reduction). However, infectious viral particles were detected
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178 on cucumbers up to 72 h p.i. Within the first 4 h p.i, viral infectivity reduces over 1 log on
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179 tomatoes and apples (1.18 and 1.27 log, respectively), while the reduction on cucumbers is only
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180 0.75 log (Table 3). The reduction in infectivity is less than 2 log at 24 h p.i on cucumbers and by

181 72 h p.i. reaches approximately 2.5 log. No infectious viral particles were detected on cucumbers

182 at 96 h p.i.

183 The median decay rate of HCoV-229E on apples and tomatoes was similar at 30%/h and 34%/h

184 respectively, while the median decay rate on cucumbers was considerably lower at 7.7%/h. The

185 median half-life of the virus on apples and tomatoes was 2.3h and 2.05h respectively and the

186 median half-life on cucumbers was 9.05h (Table 4).

187 Many factors might have contributed to the difference in viral survival on apples and tomatoes

188 compared to cucumbers, but we hypothesized that the difference in surface pH between the

189 examined produce could partly explained this observation. The surface pH of cucumbers (5.7) is

190 considerably higher than the surface pH of tomatoes and apples (4.2 and 3.9, respectively)

191 (McGlynn. 2016). For this reason, we examined viral survival on the surface of Romaine lettuce,

192 which has a surface pH close to cucumbers (5.8) (McGlynn. 2016). As shown in Figure 2,

193 similar to what has been observed for cucumbers, infectious HCoV-229E was consistently

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194 isolated at 24, 48, and 74 h p.i from the surface of lettuce. The gradual pattern of infectivity loss

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195 on lettuce resembles to what has been observed on cucumbers (Figure 2). This might indicate

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that the surface pH might play a role in viral survival at ambient temperature.
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197 Persistence of viral RNA
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198 We next set out to investigate the persistence of viral RNA on the examined produce over 72
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199 h.p.i. at ambient temperature. As demonstrated in Figure 2, no drastic reduction in viral RNA
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200 titre was observed over a 72h p.i. period. On apples, tomatoes, and cucumbers, viral RNA
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201 decreased by approximately 0.7 log, 0.5 log, and 0.3 log, respectively compared to T0,.

202 Altogether, these observations demonstrate that viral RNA is more resistant to degradation

203 compared to viral infectivity on the surface of produce.

204 Discussion

205 Currently, SARS-CoV-2 is not considered a foodborne virus, and to date, there is no conclusive

206 evidence of foodborne transmission of SARS-CoV-2. However, the traditional epidemiological

207 foodborne investigation is unlikely to be employed with COVID-19 patients. For example, it is

208 unlikely that infected people are asked to recall foods that they may have consumed during the

209 period when they became infected. Without this information, any association between SARS-
210 CoV-2 and foods cannot be made, and understanding the role of foodborne transmission remains

211 elusive. Obtaining this epidemiological information would be helpful for efficient contact-tracing

212 and source-tracking as about half of COVID-19 patients can not recall how and where they

213 contracted the virus (Tenforde, et al. 2020).

214 In this study, we used the ISO/TS 15216-1:2017 (ISO. 2017) method for the recovery of HCoV-

215 229E from the examined surfaces, and we assessed the recovery rates by plaque assay, which

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216 indicate that the recovered viruses were infectious. The recovery range that we obtained was

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217 from 4.09% to 10.77%, which is significantly higher than 1% recovery rate that is considered

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acceptable by the method. However, we speculate that the recovery efficiency would be higher if

the genetic materials were assessed instead of viral infectivity.

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220 Environmental persistence of HCoVs has been examined by different groups, who have obtained

221 contradictory results (Aboubakr, et al. 2020). One study has shown that the stability of SARS-
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222 CoV-2 and SARS-CoV-1 on dry surfaces at RT is similar, with no infectious virus being
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223 retrieved after 72h p.i. (van Doremalen, et al. 2020), while, Chin et al recovered infectious
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224 SARS-CoV-2 from plastic and stainless steel up to 7 days p.i. (Chin, et al. 2020). Keevil and

225 coworkers reported that HCoV-229E remains infectious for 5 days at RT on a range of surface

226 materials including glass and PVC, while it is rapidly inactivated on the surface of copper alloys

227 (Warnes, et al. 2015). In another study, more relevant to this work, it was shown that the

228 infectivity of HCoV-229E is completely abolished within 4 days p.i. on lettuce at 4°C (Yepiz-

229 Gomez, et al. 2013). Recently, it was demonstrated that SARS-CoV-2 remains infectious on

230 salmon at RT for 2 days (Manman. 2020). Herein, we only examined viral survival at ambient

231 temperature and we have shown the infectivity of HCoV-229E is reduced to below LOD

232 followed by 24h incubation on tomatoes and apples, and 96h on cucumbers, and lettuce.
233 At this point, we speculate that the longer survival on cucumbers, and lettuce compared to apples

234 and tomatoes could be partly explained by the difference in surface pH of these commodities.

235 The influence of pH on the stability of several coronaviruses has been studied and it has been

236 shown that in general, coronaviruses are more stable at near neutral pH as compared to acidic or

237 alkaline pH (Aboubakr, et al. 2020). As such, the near neutral surface pH of cucumbers, and

238 lettuce (5.7, and 5.8, respectively), compared to the more acidic surface pH of tomatoes and

239 apples (4.2 and 3.9, respectively), could be more suitable for the survival of HCoV-229E

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240 (McGlynn. 2016). It should also be noted that the LOD on cucumbers was lower compared to

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241 apples and tomatoes (50 PFU compared with 125 PFUs, respectively). Thus, it is possible that

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HCoV-229E remained infectious by 24 h p.i. on apples and tomatoes but the titre was below the
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243 LOD. However, the decay rate on cucumbers is considerably slower compared to apples and
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244 tomatoes (Figure 1 and Table 4), and the viral half-life on cucumbers is very close to the viral
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245 half-life on plastic (van Doremalen, et al. 2020) (9.05h and 9.04h, respectively). Further

246 investigation is needed to determine whether the surface of apples and tomatoes has some
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247 virucidal properties that may lead to a more rapid viral inactivation. Thus, our results are in
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248 accordance with the previous findings that HCoVs lose their infectivity within a few days on

249 inanimate surfaces at RT (Sizun, et al. 2000). Therefore, if produce becomes contaminated with

250 HCoVs through irrigation or contaminated hands during pre- or post-harvest, while being stored

251 at ambient temperature, the risk will be considerably reduced by the time it reaches the

252 consumers. However, if the contamination occurs at the end of the food processing chain, for

253 example by infected personnel in a restaurant setting, where the prepared food is consumed

254 within a few minutes, there is a potential risk for infection (Zelner, et al. 2020, de Wit, et al.
255 2007). Although, to date there is no evidence that ingestion of SARS-CoV-2 could lead to

256 infection.

257 The persistence of viral RNA on the studied produce for several days despite the loss of

258 infectivity, can be explained by the high environmental resilience of the coronavirus shell, which

259 protects the viral genome (Goh, et al. 2020).

260 It should be noted that our study involved experimental inoculation of fresh produce with HCoV-

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261 229E, and thus may not be fully representative of potential natural contamination. However, the

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262 infectious titre of virus used for inoculation of samples in the current study is representative of a

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worst-case scenario, if virus was found to be present on fresh produce. Herein, we attempted to
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264 address an important knowledge gap regarding the survival of human coronaviruses on fresh
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265 produce at ambient temperature, although to date, there is no conclusive evidence that food could

266 be a vehicle for SARS-CoV-2 transmission. Potential foodborne transmission poses important
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267 public health implications and may partly explain the possible recurrence of the disease and its
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268 persistent transmission. Thus, our results could support more robust decision‐making concerning
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269 risk assessment for foodborne transmission of human coronaviruses.

270 Acknowledgements
271 The authors would like to thank Dr. Brent Dixon and Dr. Franco Pagotto from the Bureau of

272 Microbial Hazards for kindly reviewing the manuscript and providing insightful comments. This

273 study is financially supported by the Bureau of Microbial Hazards, Health Canada
274 Figure legends

275 Figure 1. Persistence of infectious HCoV-229E on commonly consumed fruits and vegetables.

276 Approximately 5×104 PFU HCoV-229E (100 µl viral stock) was applied to the tested surface and

277 incubated at ambient conditions (22°C; relative humidity, 30% to 40%). Virus was extracted and

278 assayed for infectivity at various time points as described in the text. The data represent the

279 average of three independent experiments. Error bars represent standard deviation.

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280 Figure 2. Persistence of infectious HCoV-229E on Rromaine lettuce. Approximately 5×104 PFU

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281 HCoV-229E (100 µl viral stock) was applied to the tested surface and incubated at ambient

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conditions (22°C; relative humidity, 30% to 40%). Virus was extracted and assayed for
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283 infectivity at various time points as described in the text. The data represent the average of three
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284 independent experiments. Error bars represent standard deviation.

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285 Figure 3. Persistence of viral RNA on commonly consumed fruits and vegetables.

286 Approximately 2×108 RNA copies of HCoV-229E (100 µl of viral stock) was applied to the
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287 tested surface and incubated at ambient conditions (22°C; relative humidity, 30% to 40%). Virus
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288 was extracted at indicated time points and viral RNA was quantified by ddRT-PCR. The data

289 represent the average of three independent experiments. Error bars represent standard deviation.

290

291
292 References

293 Aboubakr, H. A., Sharafeldin, T. A. and Goyal, S. M. 2020. Stability of SARS-CoV-2 and other
294 coronaviruses in the environment and on common touch surfaces and the influence of climatic
295 conditions: A review. Transbound Emerg. Dis. 10.1111/tbed.13707

296 Cha, M. H., Regueiro, M. and Sandhu, D. S. 2020. Gastrointestinal and hepatic manifestations of
297 COVID-19: A comprehensive review. World J. Gastroenterol. 26, 2323-2332.

298 Chak-Yiu Lee, A., Zhang, A. J., Fuk-Woo Chan, J., Li, C., Fan, Z., Liu, F., Chen, Y., Liang, R.,
299 Sridhar, S., Cai, J. P., Kwok-Man Poon, V., Chung-Sing Chan, C., Kai-Wang To, K., Yuan, S.,
300 Zhou, J., Chu, H. and Yuen, K. Y. 2020. Oral SARS-CoV-2 inoculation establishes subclinical

of
301 respiratory infection with virus shedding in golden Syrian hamsters. Cell. Rep. Med., 100121.

ro
302 Cheung, K. S., Hung, I. F., Chan, P. P., Lung, K. C., Tso, E., Liu, R., Ng, Y. Y., Chu, M. Y.,
303 Chung, T. W., Tam, A. R., Yip, C. C., Leung, K. H., Yim-Fong Fung, A., Zhang, R. R., Lin, Y.,
304
305
-p
Cheng, H. M., Zhang, A. J., To, K. K., Chan, K. H., Yuen, K. Y. and Leung, W. K. 2020.
Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples
re
306 from the Hong Kong Cohort and Systematic Review and Meta-analysis. Gastroenterology.
307 59(1):81-95
lP

308 Chin, A. W. H., Chu, J. T. S., Perera, M. R. A., Hui, K. P. Y., Yen, H. L., Chan, M. C. W.,
309 Peiris, M. and Poon, L. L. M. 2020. Stability of SARS-CoV-2 in different environmental
na

310 conditions. Lancet Microbe 1, e10-5247(20)30003-3. Epub 2020 Apr 2.

311 de Wit, M. A., Widdowson, M. A., Vennema, H., de Bruin, E., Fernandes, T. and Koopmans, M.
ur

312 2007. Large outbreak of norovirus: the baker who should have known better. J. Infect. 55, 188-
313 193.
Jo

314 Decaro, N. and Buonavoglia, C. 2011. Canine coronavirus: not only an enteric pathogen. Vet.
315 Clin. North Am. Small Anim. Pract. 41, 1121-1132.

316 Fisher, D., Reilly, A., Kang Eng Zheng, A., Cook, A.,R. and Anderson, D.,E. 2020. Seeding of
317 outbreaks of COVID-19 by contaminated fresh and frozen food. BioRxiv.

318 Goh, G. K., Dunker, A. K., Foster, J. A. and Uversky, V. N. 2020. Shell disorder analysis
319 predicts greater resilience of the SARS-CoV-2 (COVID-19) outside the body and in body fluids.
320 Microb. Pathog. 144, 104177.

321 Guillier, L., Martin-Latil, S., Chaix, E., Thebault, A., Pavio, N., Le Poder, S., Batejat, C., Biot,
322 F., Koch, L., Schaffner, D., Sanaa, M. and Covid-19 Emergency Collective Expert Appraisal
323 Group. 2020. Modelling the inactivation of viruses from the Coronaviridae family in response to
324 temperature and relative humidity in suspensions or surfaces. Appl. Environ. Microbiol.
325 Hartman, A. L., Nambulli, S., McMillen, C. M., White, A. G., Tilston-Lunel, N. L., Albe, J. R.,
326 Cottle, E., Dunn, M. D., Frye, L. J., Gilliland, T. H., Olsen, E. L., O'Malley, K. J., Schwarz, M.
327 M., Tomko, J. A., Walker, R. C., Xia, M., Hartman, M. S., Klein, E., Scanga, C. A., Flynn, J. L.,
328 Klimstra, W. B., McElroy, A. K., Reed, D. S. and Duprex, W. P. 2020. SARS-CoV-2 infection
329 of African green monkeys results in mild respiratory disease discernible by PET/CT imaging and
330 shedding of infectious virus from both respiratory and gastrointestinal tracts. PLoS Pathog. 16,
331 e1008903.

332 Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., Zhang, L., Fan, G., Xu, J., Gu, X., Cheng,
333 Z., Yu, T., Xia, J., Wei, Y., Wu, W., Xie, X., Yin, W., Li, H., Liu, M., Xiao, Y., Gao, H., Guo,
334 L., Xie, J., Wang, G., Jiang, R., Gao, Z., Jin, Q., Wang, J. and Cao, B. 2020. Clinical features of
335 patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497-506.

of
336 ISO. 2017. EN ISO 15216-1:2017. Microbiology of the Food Chain–Horizontal Method for

ro
337 Determination of Hepatitis A Virus and Norovirus Using Real-time RT-PCR- Part1: Method for
338 Quantification. International Organization for Standardization

339
-p
Lamers, M. M., Beumer, J., van der Vaart, J., Knoops, K., Puschhof, J., Breugem, T. I., Ravelli,
re
340 R. B. G., Paul van Schayck, J., Mykytyn, A. Z., Duimel, H. Q., van Donselaar, E., Riesebosch,
341 S., Kuijpers, H. J. H., Schippers, D., van de Wetering, W. J., de Graaf, M., Koopmans, M.,
lP

342 Cuppen, E., Peters, P. J., Haagmans, B. L. and Clevers, H. 2020. SARS-CoV-2 productively
343 infects human gut enterocytes. Science. 369(6499):50-54.
na

344 Liu, P., Yang, M., Zhao, X., Guo, Y., Wang, L., Zhang, J., Lei, W., Han, W., Jiang, F., Liu, W.
345 J., Gao, G. F. and Wu, G. 2020. Cold-chain transportation in the frozen food industry may have
ur

346 caused a recurrence of COVID-19 cases in destination: Successful isolation of SARS-CoV-2

347 virus from the imported frozen cod package surface. Biosaf Health. 2(4):199-201
Jo

348 Long, A. M. and Short, S. M. 2016. Seasonal determinations of algal virus decay rates reveal
349 overwintering in a temperate freshwater pond. ISME J. 10, 1602-1612.

350 Ma, J., Qi, X., Chen, H., Li, X., Zhang, Z., Wang, H., Sun, L., Zhang, L., Guo, J., Morawska, L.,
351 Grinshpun, S. A., Biswas, P., Flagan, R. C. and Yao, M. 2020. COVID-19 patients in earlier
352 stages exhaled millions of SARS-CoV-2 per hour. Clin. Infect. Dis. ciaa1283

353 Manman, D. e. a. 2020. Long-term survival of salmon-attached SARS-CoV-2 at 4°C as a

354 potential source of transmission in seafood markets. BioRxiv.

355 McGlynn, W. 2016. The Importance of Food pH in Commercial Canning Operations. Food
356 technology fact sheet.

357 Nasheri, N., Harlow, J., Chen, A., Corneau, N. and Bidawid, S. 2020. Evaluation of Bead-Based
358 Assays in the Isolation of Foodborne Viruses from Low-Moisture Foods. J. Food Prot. 83, 388-
359 396.
360 O'Brien, B., Goodridge, L., Ronholm, J. and Nasheri, N. 2020. Exploring the potential of
361 foodborne transmission of respiratory viruses. Food Microbiol., 103709.

362 Pang, X., Ren, L., Wu, S., Ma, W., Yang, J., Di, L., Li, J., Xiao, Y., Kang, L., Du, S., Du, J.,
363 Wang, J., Li, G., Zhai, S., Chen, L., Zhou, W., Lai, S., Gao, L., Pan, Y., Wang, Q., Li, M., Wang,
364 J., Huang, Y., Wang, J., COVID-19 Field Response Group and COVID-19 Laboratory Testing
365 Group. 2020. Cold-chain food contamination as the possible origin of Covid-19 resurgence in
366 Beijing. Natl Sci Rev, nwaa264.

367 Perlman, S. and Netland, J. 2009. Coronaviruses post-SARS: update on replication and
368 pathogenesis. Nat. Rev. Microbiol. 7, 439-450.

of
369 Pusterla, N., Vin, R., Leutenegger, C. M., Mittel, L. D. and Divers, T. J. 2018. Enteric
370 coronavirus infection in adult horses. Vet. J. 231, 13-18.

ro
371 Qian, Q., Fan, L., Liu, W., Li, J., Yue, J., Wang, M., Ke, X., Yin, Y., Chen, Q. and Jiang, C.
372
373 ciaa925.
-p
2020. Direct evidence of active SARS-CoV-2 replication in the intestine. Clin. Infect. Dis.
re
374 Roxanne Liu, D.S., August 13, 2020. Traces of coronavirus found in frozen chicken wings,
375 shrimp packaging in China, Global News. Available at
lP

376 https://globalnews.ca/news/7271588/coronavirus-traces-frozen-chicken-shrimp-packaging/
na

377 Scaldaferri, F., Ianiro, G., Privitera, G., Lopetuso, L. R., Vetrone, L. M., Petito, V., Pugliese, D.,
378 Neri, M., Cammarota, G., Ringel, Y., Costamagna, G., Gasbarrini, A., Boskoski, I. and Armuzzi,
379 A. 2020. The Thrilling Journey of SARS-CoV-2 into the Intestine: From Pathogenesis to Future
ur

380 Clinical Implications. Inflamm. Bowel Dis.

Jo

381 Sizun, J., Yu, M. W. and Talbot, P. J. 2000. Survival of human coronaviruses 229E and OC43 in
382 suspension and after drying onsurfaces: a possible source ofhospital-acquired infections. J. Hosp.
383 Infect. 46, 55-60.

384 Tenforde, M. W., Billig Rose, E., Lindsell, C. J., Shapiro, N. I., Files, D. C., Gibbs, K. W.,
385 Prekker, M. E., Steingrub, J. S., Smithline, H. A., Gong, M. N., Aboodi, M. S., Exline, M. C.,
386 Henning, D. J., Wilson, J. G., Khan, A., Qadir, N., Stubblefield, W. B., Patel, M. M., Self, W. H.,
387 Feldstein, L. R. and CDC COVID-19 Response Team. 2020. Characteristics of Adult Outpatients
388 and Inpatients with COVID-19 - 11 Academic Medical Centers, United States, March-May
389 2020. MMWR Morb. Mortal. Wkly. Rep. 69, 841-846.

390 van Doremalen, N., Bushmaker, T., Morris, D. H., Holbrook, M. G., Gamble, A., Williamson, B.
391 N., Tamin, A., Harcourt, J. L., Thornburg, N. J., Gerber, S. I., Lloyd-Smith, J. O., de Wit, E. and
392 Munster, V. J. 2020. Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-
393 CoV-1. N. Engl. J. Med. 382, 1564-1567.
394 Vijgen, L., Keyaerts, E., Moes, E., Maes, P., Duson, G. and Van Ranst, M. 2005. Development
395 of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of
396 human coronaviruses OC43 and 229E. J. Clin. Microbiol. 43, 5452-5456.

397 Wang, Q., Vlasova, A. N., Kenney, S. P. and Saif, L. J. 2019. Emerging and re-emerging
398 coronaviruses in pigs. Curr. Opin. Virol. 34, 39-49.

399 Wang, W., Xu, Y., Gao, R., Lu, R., Han, K., Wu, G. and Tan, W. 2020. Detection of SARS-
400 CoV-2 in Different Types of Clinical Specimens. JAMA.

401 Warnes, S. L., Little, Z. R. and Keevil, C. W. 2015. Human Coronavirus 229E Remains
402 Infectious on Common Touch Surface Materials. mBio 6, e01697-15.

of
403 Wolfel, R., Corman, V. M., Guggemos, W., Seilmaier, M., Zange, S., Muller, M. A., Niemeyer,

ro
404 D., Jones, T. C., Vollmar, P., Rothe, C., Hoelscher, M., Bleicker, T., Brunink, S., Schneider, J.,
405 Ehmann, R., Zwirglmaier, K., Drosten, C. and Wendtner, C. 2020. Virological assessment of
406

407
-p
hospitalized patients with COVID-2019. Nature 581, 465-469.

Xiao, F., Sun, J., Xu, Y., Li, F., Huang, X., Li, H., Zhao, J., Huang, J. and Zhao, J. 2020.
re
408 Infectious SARS-CoV-2 in Feces of Patient with Severe COVID-19. Emerg. Infect. Dis. 26,
409 1920-1922.
lP

410 Xu, H., Zhong, L., Deng, J., Peng, J., Dan, H., Zeng, X., Li, T. and Chen, Q. 2020a. High
na

411 expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int. J. Oral
412 Sci. 12, 8-020-0074-x.
ur

413 Xu, Y., Li, X., Zhu, B., Liang, H., Fang, C., Gong, Y., Guo, Q., Sun, X., Zhao, D., Shen, J.,
414 Zhang, H., Liu, H., Xia, H., Tang, J., Zhang, K. and Gong, S. 2020b. Characteristics of pediatric
Jo

415 SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding. Nat. Med. 26,
416 502-505.

417 Yepiz-Gomez, M. S., Gerba, C. P. and Bright, K. R. 2013. Survival of Respiratory Viruses on
418 Fresh Produce. Food Environ. Virol.5(3):150–6.

419 Yusha, Z., 2020. China’s CDC experts investigate Xinfadi market three times, announce
420 groundbreaking virus tracing discovery, Global Times. Available at
421 https://www.globaltimes.cn/content/1192146.shtml

422 Zelner, J., Adams, C., Havumaki, J. and Lopman, B. 2020. Understanding the Importance of
423 Contact Heterogeneity and Variable Infectiousness in the Dynamics of a Large Norovirus
424 Outbreak. Clin. Infect. Dis. 70, 493-500.

425 Zhou, J., Li, C., Liu, X., Chiu, M. C., Zhao, X., Wang, D., Wei, Y., Lee, A., Zhang, A. J., Chu,
426 H., Cai, J. P., Yip, C. C., Chan, I. H., Wong, K. K., Tsang, O. T., Chan, K. H., Chan, J. F., To, K.
427 K., Chen, H. and Yuen, K. Y. 2020. Infection of bat and human intestinal organoids by SARS-
428 CoV-2. Nat. Med. 26(7):1077-1083.

429 Zhou, J., Li, C., Zhao, G., Chu, H., Wang, D., Yan, H. H., Poon, V. K., Wen, L., Wong, B. H.,
430 Zhao, X., Chiu, M. C., Yang, D., Wang, Y., Au-Yeung, R. K. H., Chan, I. H., Sun, S., Chan, J.
431 F., To, K. K., Memish, Z. A., Corman, V. M., Drosten, C., Hung, I. F., Zhou, Y., Leung, S. Y.
432 and Yuen, K. Y. 2017. Human intestinal tract serves as an alternative infection route for Middle
433 East respiratory syndrome coronavirus. Sci. Adv. 3, eaao4966.

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435 Table 1. Recovered viral titre at T0 and recovery rate in percentage for each produce type. The
436 results are the mean of 3 independent experiments.
Produce Titer at T0 Recovery rate
(PFU/mL) (%)
Apple 1.45E+03 5.81
Tomato 2.69E+03 10.77
Cucumber 1.20E+03 4.09
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439 Table 2. Detection of HCoV-229E on the surface of different produce. Samples were inoculated

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440 with 104 to 101 PFU of HCoV-229E and examined by plaque assay at T0. ND is not detected.
Produce Viral Inoculum (PFU)
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Apple 3/3 3/3 3/3 3/3 3/3 ND ND
Tomato 3/3 3/3 3/3 3/3 3/3 2/3 ND
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Cucumber 3/3 3/3 3/3 3/3 3/3 3/3 ND

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442 Table 3. Log reduction in viral titre compared to T0. The results are the mean of 3 independent
443 experiments ± Standard Deviation.

Time point Apples Tomatoes Cucumbers

0.5h 0.09±0.01 0.09±0.05 0.10±0.01
1h 0.23±0.06 0.14±0.04 0.33±0.11
2h 0.90±0.12 0.68±0.05 0.38±0.11
4h 1.08±0.18 1.05±0.02 0.76±0.01
6h 1.27±0.08 1.18±0.06 0.79±0.04
16h 2.40±0.33 2.37±0.09 1.26±0.06
24h 3.16 3.43 1.92±0.15
48h 3.16 3.43 2.09±0.16

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72h 3.16 3.43 2.48±0.035
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446 Table 4. Decay rate (DR) in percentage and viral half-life (HL) in hours (h) on each produce
447 type. The results are the median of 3 independent experiments ± Standard Deviation.
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DR (%) HL (h)
Apple 30±0.25 2.3±0.02
Tomato 34±0.1 2.05±0.06
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Cucumber 7.7±0.6 9.05±0.75

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450 Figure 1

10000

1000
Viral Titre (PFU/mL)

100 Apples
Tomatoes
Cucumbers
10

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454 Figure 2.

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459 Figure 3.

1.00E+09

1.00E+08

1.00E+07

1.00E+06
RNA Copies

1.00E+05
Apples
1.00E+04
Tomatoes
1.00E+03
Cucumbers

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1.00E+02

1.00E+01

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1.00E+00
0h 0.5h 1h 2h 4h 6h 16h 24h 48h 72h

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• HCoV-229E remains infectious on the surface of apples and tomatoes for 16h
• HCoV-229E remains infectious on the surface of cucumbers and lettuce for 72h
• Viral infectivity decay rate is significantly slower on cucumbers compared with apples and
tomatoes
• Viral RNA remains stable on all produce with no significant decrease

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The authors declare no conflict of interest.

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